Increased photosynthetic efficiencies in genotypes with greater proline level and in crops treated with proline under water deficit have been reported in recent years, but the biochemical and molecular mechanisms of this process are still not known. We examined photosystem II (PSII) activity, photosynthetic enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), phosphoenolpyruvate carboxylase (PEPc), rubisco activase (RCA), and chlorophyll metabolic enzymes, magnesium chelatase (Mg-CHLI), and chlorophyllase (Chlase), which would be the primary targets of exogenous proline to provide photosynthetic protection to plants under PEG-induced short-term water deficit. Two maize genotypes W23/M14 with greater proline content and Safak with low proline content were hydroponically grown for 21-23 days, and then the seedlings were subjected to water deficit (- 0.75 MPa) induced by PEG(6000) for 0, 4, and 8 h. Before the seedlings were exposed to the water deficit, proline (1 mM) was applied to the rooting medium of the Safak genotype for 2 days. The time course effects of the applications showed that exogenous proline significantly enhanced PSII efficiency, PEPc activity, rubisco activity, and the relative expression levels of PEPc, rubisco large subunit, rubisco small subunit, and RCA genes at 0, 4, and 8 h. The W23/M14 genotype had higher rubisco quantity than the Safak genotype at all time periods. Proline pre-treatment under the stress-free and PEG conditions reduced the activity of Chlase and the gene expressions of Chlase, while it enhanced Mg-CHLI gene expression at 0, 4, and 8 h. Taken together, the results indicated that the primary target of proline-stimulated signaling in maize seedlings exposed to short-term severe water deficit may be to induce PSII efficiency, activities of carbon dioxide fixation enzymes and chlorophyll metabolism and mitigate chlorophyll degradation.